CD4O, a 50kD glycoprotein expressed on all B cells, has been shown to play an important role in B cell development and activation. Anti CD40 monoclonal antibodies (mAb) have been shown to stimulate resting B cells to proliferate and induce Ig class switch to IgB synthesis in the presence of IL-4. The ligand for CD4O has been identified to be a glycoprotein of 39kD. Mutations or deletions of this CD40 ligand have been shown to be the cause of X-linked immunodeficiency associated with either normal or excessive IgM. Both the sequences of cDNA encoding for CD40 and its ligand have been determined. The CD40 system is readily amendable for further analysis. Recently, signalling through CD40 is shown to be distinct from that through surface Ig. Protein tyrosine phosphorylation and dephosphorylation during the early phase of engaging CD40 with anti CD4O mAb define this B cell activation pathway via CD40. Its association with a unique 28 kD tyrosine phosphorylated protein has been established. In this proposal, the CD4O activation pathway will be investigated with the following specific aims: 1) to define the molecules associated with CD4O with specific emphasis to identify associated proteins with a motif characteristic of proteins interacting with src protein tyrosine kinases (PTK), 2) to define the phosphoproteins associated with the CD40 complex, some of which are postulated to be members of the src family, and dephosphorylated and rephosphorylated rapidly during the early phase of CD40 signal transduction, 3) to explore the role of phosphatases in the CD40 signalling pathway with a special emphasis on the role of CD45 in this process, 4) to identify the structure of the 28 kD protein which is tyrosine-phosphorylated within seconds after CD40 engagement by anti CD4O mAb, 5) to explore the role of the recently described B cell cytoplasmic protein tyrosine kinase, which is deficient in human X-linked agammaglobulinemia in CD4O signal transduction, and 6) to determine whether the 21kD phosphoprotein associated with the CD40 complex is indeed p21ras. Recent studies have shown that engagement of CD4O in certain B cell tumor systems inhibit tumor growth. Thus the understanding of the CD4O signalling pathway will provide new insights in B cell activation as well as therapeutic implications.
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